A single-use glass microcapillary (FemtoTips, Eppendorf) was connected to a pump (FemtoJet 5247 or FemtoJet 4i, Eppendorf), and the movement of the microcapillary was controlled by a micromanipulator (TransferMan 4r, Eppendorf). The micromanipulator was mounted on the body of the microscope. A pipette tip (Microloader, Eppendorf) was used to add 1 μl of pure water into the tip of the microcapillary. A constant inner pressure of 60 hPa against capillary force was applied inside the microcapillary by the pump. The focal plane was kept constant at the position of the upper surface of the microchamber array during the recovery experiment. As we lowered the microcapillary toward the droplet of interest, a latent image of the microcapillary was first observed via eyepieces under bright field, which revealed the approximate location of the microcapillary. A pressure drop (down to 0 hPa, kept for 1 s) was triggered when the tip was inserted into the target droplet, resulting in an instant suction for the femtoliter solution. The large concentration difference inside and outside the microcapillary facilitates a quick diffusion of the contents into the preloaded pure water. A fast release of the recovered contents into a PCR tube preloaded with PCR reagents (with the same primer set as the one used in the preparation of the linear template DNA) was triggered by touching the tip of the microcapillary with the inner wall of the PCR tube. The capillary force was decreased in inverse proportion to the diameter of the tip, and the constant inner pressure pushed the liquid out of the microcapillary as a result of disruption of a force balance. Because of the large dilution factor (>108-fold) of the contents of the CFPS solution, the following PCR was not affected by the impurities. Two rounds of normal PCR in a volume of 25 μl, each consisting of 30 cycles, were able to amplify a single DNA molecule to a level exceeding the detection limit of agarose gel electrophoresis. The first-round PCR solution (0.5 μl) was added into the second-round PCR without DNA purification. The amplicons were purified for Sanger sequencing or they may be used directly as the template DNA of CFPS.

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